The present invention relates to template devices and more particularly, the invention relates to a template for forming multiple-concentration gel tracks in an electrophoretic gel bed.
Gel electrophoresis is a technique that is currently used in the separation of proteins, lipoproteins and DNA. More recently this technique has been demonstrated to be useful in the separation and characterization of bacteriophages, [Serwer, P. and Pichler, M. E., Electrophoresis of Bacteriophage T7 and T7 Capsids in Agarose Gels, 28 Journal of Virology 917 (1980)]. Electrophoretic mobilities in agarose gels are determined by average electrical surface charge density (.rho.) and particle size. Assuming particles to be spherical, the mobility in the absence of a gel is more dependent on .rho. than size [Shaw, Electrophoresis, Academic Press (1969)]. Therefore, particles of similar charge but different radius are not separated in the absence of a gel. To accentuate the size differential of particles, electrophoresis is conducted in agarose gels of varying concentrations. As the concentration of agarose is increased the mobility of particles decreases during electrophoresis. Furthermore, the mobilities of the larger particles decrease more than mobilities of smaller particles. This is called differential sieving. It is this differential sieving which induces the fractionation and separation of a mixture of particles of equals .rho., such as DNA or some protein, into discrete units.
On the basis that the logarithm of electrophoretic mobility is a linear function of gel concentration, the absolute electrophoretic mobility of a particle in the absence of agarose can be determined by extrapolating the log (mobility) versus agarose concentration line to 0% agarose. The slope of the plot of mobility against gel concentration can also be used to help determine the size of particles. A problem the experimenter faces is increasing the accuracy of these extrapolations by using techniques that minimize variations in temperature and voltage gradients among the gels and prevent shrinkage of gels during electrophoresis. While extrapolation to a theoretical value becomes more accurate as the concentration of agarose is decreased, the practical aspects of gel electrophoresis become increasingly more difficult. As the agarose concentration is decreased the gels become increasingly fragile and, therefore, difficult to load and to stain for visualization.
Various procedures heretofore have been employed to develop a more precise electrophoretic mobility versus gel concentration pattern. For example, the FANGMAN BOX sold by Bethesda Research Laboratories is used to encase a single running gel within a higher concentration of agarose frame gel. The purpose of the frame gel is to provide structural stability to the less viscous running gel. Low concentration running gels on the order of 0.1% w/v or greater are routinely employed in this electrophoretic technique, thereby extending the lower limits of the experimental line on a log (mobility) versus gel concentration plot. However, as a drawback in performing the experiment, the experimenter must employ several FANGMAN BOXES each having a different concentration of running gel or sequentially perform the electrophoresis in the same box changing the gel concentration after each run. Either method introduces variable error in the experiment involving temperature, humidity, and voltage gradients which cannot be controlled or normalized from box to box or run to run.
In efforts to normalize the random error imposed by ambient conditions from batch to batch, experimenters have tried running multiple concentration gels in the same apparatus. A vertical apparatus for agarose gel electrophoresis is described by Johnson, P. H. and Grossman, L. I., 16 Biochemistry 4217 (1977). The apparatus consists of a vertical gel bed equipped with plastic partitions separating the gel bed into several frames, each frame of which is filled with a gel of varying agarose concentrations. A drawback to this compartmentalization system is that gel concentrations less than 0.4% are not feasible in that there is slippage of gels from between the plates holding them, sample wells break during comb removal, there is difficulty in handling the gels for staining, and there is shrinkage of the gels during electrophoresis. Although the shrinkage problem and to a limited extent the well breakage problem can be overcome by running the electrophoresis in a horizontal mode, the other problems still exist so as to offer imprecise data.
From the foregoing, it will be appreciated that those techniques which encase the running gel within a frame gel for stability do not permit the use of multiple gels, and therefore do not normalize errors induced by ambient conditions. Moreover, those techniques for running several gels in a single apparatus are limited to relatively high gel concentration (.gtoreq.0.4% w/v) such that an accurate extrapolation to absolute electrophoretic mobility is difficult to achieve.
As a result of the shortcomings of the prior art, typified by the above, there has developed and continues to exist a substantial need for a means to develop multiple concentration gel tracks in a single gel bed which will also accommodate low concentration gels. Despite this recognized need, such a device has heretofore been unavailable.